Chapter 5

Chapter 5: Expression
Expression
Learning Objectives: Describe a generalized replication cycle for each of the seven virus genome types.
Explain how the pattern of gene expression of a

virus is determined by the structure of the virus genome and how it is replicated. Understand the role of post-transcriptional events in controlling virus gene expression.
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Expression of Genetic Information

The course of virus replication is determined by
tight control of gene expression.

There are fundamental differences in the control of these processes in prokaryotic and eukaryotic cells. Cells have varied and complex mechanisms for controlling gene expression. Viruses have had to achieve highly specific quantitative, temporal, and spatial control of expression with limited genetic resources.
Expression of Genetic Information

Powerful positive and negative signals which promote or repress gene expression Highly compressed genomes in which overlapping
reading frames are commonplace

Control signals which are frequently nested within other genes Several strategies designed to create multiple polypeptides from a single messenger RNA
Control of Prokaryote Gene Expression

Initiation of transcription is negatively regulated by synthesis of trans-acting repressor proteins, which bind to operator sequences upstream of protein coding sequences. Collections of metabolically related genes are grouped together and co-ordinately controlled as 'operons', typically producing a polycistronic mRNA. Transcription is regulated by mechanisms such as antitermination and modifications of RNA polymerase. At a post-transcriptional level, gene expression is also regulated by control of translation.
Control of Gene Expression in Bacteriophage l

Phage l was discovered by Esther Lederberg in 1949.
Experiments at the Pasteur Institute by Andr Lwoff in 1950 showed that some strains of bacteria, when irradiated with ultraviolet light, stopped growing and lysed, releasing a crop of bacteriophage particles.
The cells of some bacterial strains carried a bacteriophage in a dormant form, known as a prophage, and that the phage could be made to alternate between the lysogenic (non-productive) and lytic (productive) growth cycles.
A Simplified Genetic Map of l
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Gene Expression in l (1)

PL is the promoter responsible for transcription of the left-hand side of the l genome, including N and cIII. OL is a short non-coding region of the phage genome
(approximately 50 bp) which lies between the cI and N

genes next to PL. PR is the promoter responsible for transcription of the right-hand side of the l genome, including cro, cII, and the genes encoding the structural proteins.

OR is a short non-coding region of the phage genome
(approximately 50 bp) which lies between the cI and cro

genes next to PR. cI is transcribed from its own promoter and encodes a
repressor protein of 236 amino acids which binds to OR,

preventing transcription of cro but allowing transcription of cI, and to OL, preventing transcription of N and the
other genes in the left-hand end of the genome.

cII and cIII encode activator proteins which bind to the genome, enhancing the transcription of the cI gene.

cro encodes a 66 amino acid protein which binds to
OR, blocking binding of the repressor to this site.

N encodes an antiterminator protein which acts as an alternative (rho) factor for host cell RNA polymerase, modifying its activity and permitting extensive transcription from PL and PR.
Q is an antiterminator similar to N, but only permits

extended transcription from PR.
Gene Expression in l
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Gene Expression in l
Elsevier, 2005.
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Control of Eukaryote Gene Expression

Control of gene expression in eukaryotic cells is more
complex than in prokaryotic cells, a 'multilayered'

approach. DNA in eukaryotic cells has an elaborate structure,
forming complicated and dynamic complexes with

numerous proteins to form chromatin. Transcriptionally active DNA is also hypomethylated
compared with the frequency of methylation in

transcriptionally quiescent regions of the genome.
Elsevier, 2005.
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Initiation of transcription is influenced by sequences upstream of the transcription start site, which are binding sites for families of highly specific DNAbinding proteins, 'transcription factors'. Immediately upstream of the transcription start site is a relatively short region known as the promoter. Sequences further upstream from the promoter influence the efficiency with which transcription complexes form. The rate of initiation depends on the combination of transcription factors bound to these transcription enhancers.
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The stability of eukaryotic mRNAs varies considerably, some having comparatively long halflives in the cell (many hours). The half-lives of others which encode regulatory proteins may be very short (a few minutes). Gene expression is also regulated by differential splicing of heterogeneous (heavy) nuclear RNA (hnRNA) precursors in the nucleus. In eukaryotic cells, control is also exercised during export of RNA from the nucleus to the cytoplasm. The efficiency with which different mRNAs are translated varies greatly.
Genome Coding Strategies: Class I: Double-Stranded DNA

Can be divided into two groups: replication is exclusively nuclear (e.g. Adenoviridae, Polyomaviridae, Herpesviridae) replication occurs in the cytoplasm (Poxviridae)
Since their genomes all resemble double-stranded cellular DNA, they are essentially transcribed by the same mechanisms as cellular genes However, there are profound differences between them relating to the degree to which each family is reliant on the host cell machinery.
Polyomaviruses and Papillomaviruses

Polyomaviruses are heavily dependent on cellular machinery both for replication and gene expression Polyomaviruses encode trans-acting factors (Tantigens) which stimulate transcription (and genome replication) Papillomaviruses in particular are dependent on the cell for replication, which only occurs in terminally differentiated keratinocytes.
Adenoviruses
Adenoviruses are also heavily dependent on the cellular apparatus for transcription, but possess various mechanisms that regulate virus gene expression. These include trans-acting transcriptional activators such as the E1A protein, and posttranscriptional regulation of expression. Adenovirus infection of cells is divided into two stages, early and late, the latter commencing at the time when genome replication occurs.
Elsevier, 2005.
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Herpesviruses
These viruses are less reliant on cellular enzymes than most other viruses in this class. They encode many enzymes involved in DNA metabolism (e.g. thymidine kinase) and several trans-acting factors that regulate the temporal expression of virus genes, controlling the phases of infection. Transcription of the large, complex genome is sequentially regulated in a cascade fashion.
Herpesvirus Gene Expression
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Poxviruses
Genome replication and gene expression in poxviruses are almost independent of cellular mechanisms (except for host cell ribosomes). Poxvirus genomes encode numerous enzymes involved in DNA metabolism, virus gene transcription, and post-transcriptional modification of mRNAs. Many of these enzymes are packaged within the virus particle (which contains >100 proteins), enabling transcription and replication of the genome to occur in the cytoplasm almost totally under the control of the virus.
Poxviruses
Gene expression is carried out by virus enzymes associated with the core of the particle and is divided into two rather indistinct phases: Early genes: about 50% of the poxvirus genome, expressed before genome replication inside a partially uncoated core particle resulting in the production of 5' capped, 3' polyadenylated but unspliced mRNAs. Late genes: expressed after genome replication in the cytoplasm, but their expression is also dependent on virus-encoded rather than on cellular transcription proteins.
Genome Coding Strategies: Class II: Single-Stranded DNA

Both the autonomous and the helper virusdependent parvoviruses are highly reliant on external assistance for gene expression and genome replication. The members of the replication-defective Dependovirus genus of the Parvoviridae are entirely dependent on adenovirus or herpesvirus superinfection for helper functions essential for replication. The adenovirus genes required as helpers are the early, transcriptional regulatory genes such as E1A rather than late structural genes.
Genome Coding Strategies: Class II: SingleStranded DNA
Elsevier, 2005.
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Genome Coding Strategies: Class II: Single-Stranded DNA

The Geminiviridae also fall into this class of genomes. The expression of their genomes is quite different from that of parvoviruses, but nevertheless still relies heavily on host cell functions. There are open reading frames in both orientations in the virus DNA, which means that both (+) and ( )sense strands are transcribed during infection.
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Genome Coding Strategies: Class III: Double-Stranded RNA

All viruses with RNA genomes differ fundamentally
from their host cells.

Reoviruses have segmented genomes and replicate in the cytoplasm of the host cell. Early in infection, transcription of the ds RNA genome segments by virus-specific transcriptase activity occurs inside partially uncoated sub-virus particles.
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Genome Coding Strategies: Class III: DoubleStranded RNA
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Genome Coding Strategies: Class III: Double-Stranded RNA

Primary transcription results in capped transcripts that are not polyadenylated. The various genome segments are transcribed/translated at different frequencies. RNA is transcribed conservatively, resulting in synthesis of (+)sense mRNAs, which are capped inside the core. Secondary transcription occurs later in infection inside new particles produced in infected cells and results in uncapped, non-polyadenylated transcripts. The genome is replicated in a conservative fashion.
Genome Coding Strategies: Class IV: Single-Stranded (+)Sense RNA

These virus genomes act as messenger RNAs and are themselves translated immediately after infection of the host cell. This class of genomes displays a very diverse range of strategies for controlling gene expression and genome replication.
There are two main strategies of gene expression.
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Production of a polyprotein encompassing the whole of the virus, which is cleaved by proteases to produce precursor and mature polypeptides.
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Production of subgenomic mRNAs, resulting from two or more rounds of translation of the genome.
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Genome Coding Strategies: Class V: Single-Stranded (-)Sense RNA

The genomes of these viruses may be either segmented or non-segmented. The first step in the replication of segmented orthomyxovirus genomes is transcription of the ( )sense vRNA by the virion-associated RNAdependent RNA polymerase to produce monocistronic mRNAs, which also serve as the template for genome replication.
Packaging of a virus-specific transcriptase/replicase within the virus nucleocapsid is essential.

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Viruses with non-segmented genomes also produce monocistronic mRNAs. These are produced individually by a stop and start mechanism of transcription regulated by the conserved intergenic sequences present between each of the virus genes. Splicing mechanisms cannot be used because these viruses replicate in the cytoplasm.
The ratio of different proteins is regulated both during transcription and afterwards.
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Paramyxovirus Gene Expression
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Class VI: Single-Stranded (+)Sense RNA with a DNA Intermediate

The retrovirus RNA genome forms a template for reverse transcription to DNA - these are the only (+)sense RNA viruses whose genome does not serve as mRNA on entering the host cell. Once integrated into the host cell genome, the DNA provirus is under the control of the host cell and is transcribed like cellular genes. Some retroviruses have evolved a number of transcriptional and post-transcriptional mechanisms that allow them to control the expression of their genetic information.
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Class VII: Double-Stranded DNA with an RNA Intermediate

Expression of Hepadnavirus genomes is complex and relatively poorly understood. The hepadnaviruses contain a number of overlapping reading frames clearly designed to squeeze as much coding information as possible into a compact genome. The X gene encodes a transcriptional trans-activator believed to be analogous to the HTLV tax protein. At least two mRNAs are produced from independent promoters, each of which encodes several proteins and the larger of which is also the template for reverse transcription during the formation of the virus particle.
Transcriptional Control of Gene Expression

The SV40 genome encodes two T-antigens ('tumour antigens') known as large T-antigen and small Tantigen after the sizes of the proteins. Replication of the double-stranded DNA genome of SV40 occurs in the nucleus of the host cell. Transcription of the genome is carried out by host cell RNA polymerase II but large T-antigen regulates transcription of the virus genome. Small T-antigen is not essential for virus replication, but allows virus DNA to accumulate in the nucleus. Both proteins contain 'nuclear localization signals' which results in their accumulation in the nucleus.
The SV40 Genome
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SV40 Gene Expression
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Retrovirus Gene Expression

The presence of numerous binding sites for cellular transcription factors in the long terminal repeats (LTRs) of the provirus have been analysed by 'DNAse I footprinting' and 'gel-shift' assays. The 'distal' elements (such as NFkB and SP1 bindingsites) and 'proximal' elements (such as the TATA box) make up a transcription promoter in the U3 region of the LTR. The basal activity of these promoters is relatively weak, and results in only limited transcription of the provirus genome by RNA polymerase II.
DNA Binding proteins and Retrovirus LTRs
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Retrovirus Gene Expression
Both HTLV and HIV encode proteins which are trans-acting positive regulators of transcription, the tax protein of HTLV and the HIV tat protein.
These proteins act to increase transcription from

the virus LTR to at least 50-100 times that of the basal rate of the promoter. Neither the tax nor the tat protein bind directly to their respective LTRs.
Gene Expression in HTLV and HIV
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Control of HTLV Gene Expression

HTLV tax binds directly to three 21bp GC-rich CRE
(cyclic AMP response element) sequences in the virus

LTR. tax also forms protein-protein interactions with cellular
transcription factors, e.g. p105, a precursor of NF-kB.

tax enhances dimerization of 'bZIP' proteins that bind to DNA via a basic domain-leucine zipper region.
Dimerization of these proteins is required for DNA

binding and for transcriptional activation.
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Control of HIV Gene Expression

The HIV tat protein binds to a stem-loop structure at the 5' end of HIV mRNAs, known as TAR (Trans-Activation Response) element. In the absence of Tat, initiation from the LTR is efficient, but transcription is impaired because the promoter forms a poorly processive polymerase complex. In the presence of tat bound to TAR, a different transcription initiation complex forms which is competent to complete transcription of the HIV genome. The HTLV tax and HIV tat proteins are positive regulators of the basal promoter in the provirus LTR and are under the control of the virus.
Control of HTLV/HIV Gene Expression
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Control of Transcription
Even some of the simplest virus genomes, such as SV40, encode proteins that regulate their transcription. Many virus genomes encode trans-acting factors that modify the cellular transcription apparatus. Examples of this include HTLV and HIV, but also the X protein of hepadnaviruses, rep protein of parvoviruses, E1A protein of adenoviruses and the immediate early proteins of herpesviruses. The expression of RNA virus genomes is similarly tightly controlled, but this process is carried out by virus-encoded transcriptases.
Splicing
Many DNA viruses that replicate in the nucleus encode mRNAs which must be spliced by cellular mechanisms. Splicing requires the processing of mRNAs by nuclear apparatus before they are transported into the cytoplasm for translation. Several virus families have taken advantage of this capacity of their host cells to compress more genetic information into their genomes, e.g. parvoviruses and polyomaviruses. In contrast, the large genetic capacity of herpesviruses makes it possible for these viruses to produce mostly unspliced monocistronic mRNAs.
Adenovirus Gene Expression

Families' of adenovirus genes are expressed via
differential splicing of precursor hnRNA transcripts.

The first proteins to be expressed, E1A and E1B, are encoded by a transcriptional unit on the r-strand at the extreme left-hand end of the adenovirus genome. These proteins are primarily transcriptional trans-
regulatory proteins comparable to the tax and tat

proteins.
Adenovirus Gene Expression
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Adenovirus E1A
Five related E1A polypeptides are produced from the same reading frame and have the same amino and carboxy termini. The 289 and 243 amino acid peptides are transcriptional activators. Although these proteins activate transcription from all the early adenovirus promoters, they are also 'promiscuous', activating most RNA polymerase IIresponsive promoters that contain a TATA box. There are no obvious common sequences present in all of these promoters and there is no evidence that the E1A proteins bind directly to DNA.
Expression of Adenovirus E1A
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Adenovirus E1A
E1A proteins from different adenovirus serotypes contain three conserved domains and interact with many other cellular proteins. E1A can both activate and repress transcription.
Synthesis of E1A starts a cascade of transcriptional activation by turning on transcription of the other adenovirus early genes, E1B, E2, E3, and E4. After the virus genome has been replicated, this cascade results in transcription of the late genes encoding the structural proteins.
Adenovirus E1A
Transcription of the E1A is a balanced, selfregulating system. The immediate early genes of DNA viruses typically have strong enhancer elements upstream of their promoters. The immediate early proteins are transcriptional activators that turn on expression of other virus genes. Although E1A trans-activates its own promoter, the protein represses the function of the upstream enhancer element and at high concentrations, also down-regulates its own expression.
Adenovirus E1A
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Post-Transcriptional Control of Expression by Adenoviruses

The Adenovirus VA (virus-associated) genes encode two small (~160 nt) RNAs transcribed from the rstrand of the genome by RNA polymerase III during the late phase of virus replication. Both VA RNA I and VA RNA II have a high degree of secondary structure and neither molecule encodes any polypeptide and accumulate to high levels in the cytoplasm of adenovirus-infected cells.
The way in which these two RNAs act is not completely understood, but their net effect is to boost the synthesis of adenovirus late proteins.
Post-Transcriptional Control of Expression by Adenoviruses

Interferons activate a cellular protein kinase known as PKR that inhibits initiation of translation. VA RNA I binds to PKR, preventing its activity. The effects of interferons on the cell are generalized and result in inhibition of the translation of both cellular and virus mRNAs. The VA RNAs may be able to promote selectively the translation of adenovirus mRNAs at the expense of cellular mRNAs whose translation remains inhibited.
Translation Efficiency
The efficiency with which different mRNAs are translated is determined by the nucleotide sequence surrounding the AUG translation initiation codon. The most favourable sequence for initiation is GCC(A/G)CCAUGGG. A number of viruses use variations of this sequence to regulate the amounts of protein synthesized from a single mRNA. Examples of this are the tax and rex proteins of HTLV, which are encoded by overlapping reading frames in the same doubly spliced 2.1 kb mRNA - the 'leaky scanning' mechanism.
Translation Initiation
Picornavirus genomes have long non-coding regions at their 5' ends. These sequences are involved in the replication and possibly packaging of the virus genome. Translation of most mRNAs is initiated when ribosomes recognize the 5' end of the mRNA and scan the sequence until they reach an AUG initiation codon. The 5' end of the Picornavirus genome is not capped, but is modified by the addition of the VPg protein. There are multiple AUG codons in the 5' NCR upstream of the start of the polyprotein coding sequences which are not recognized by ribosomes.
The Internal Ribosome Entry Site (IRES)

In picornavirus-infected cells, a virus protease cleaves the 220 kDa 'cap-binding complex' (CBC) which binds the cap structure at the 5' end of mRNA during initiation of translation. Rather than scanning along picornavirus RNA from the 5' end, ribosomes bind to the RNA via an internal ribosomal entry site (IRES) and begin translation internally. Few cellular mRNAs utilize this mechanism but it has been shown to be used by a variety of viruses, including picornaviruses, hepatitis C virus, coronaviruses and flaviviruses.
Decoding the Genome

Many viruses compress their genetic information by encoding different polypeptides in overlapping reading frames. If each polypeptide is expressed from a monocistronic mRNA transcribed from its own promoter, the additional cis-acting sequences required to control expression might cancel out any advantage gained. There is also the problem of co-ordinately regulating the transcription and translation of multiple different messages. It is therefore desirable to express several polypeptides from a single RNA transcript.
Ribosomal Frameshifting
Ribosomal frameshifting was first discovered in viruses, but also occurs in prokaryotic and eukaryotic cells. Retrovirus genomes are transcribed to produce at least two 5' capped, 3' polyadenylated mRNAs. A long, unspliced transcript encodes the gag, pro, and pol genes and also forms the genomic RNA packaged into virions. The problem is how to express three different proteins from one long transcript.
In some cases (e.g. HTLV), the gag, pro, and pol genes occupy three different reading frames, while in others (e.g. HIV), the protease (pro) gene forms an extension at the 5' end of the pol gene. In HIV, the protease and polymerase are expressed as a polyprotein which is cleaved into the mature proteins in a process similar to the cleavage of picornavirus polyproteins.
At the boundary between each of the three genes there is a sequence which consists of a tract of reiterated nucleotides, such as UUUAAAC. Most ribosomes encountering this sequence translate it and continue along the transcript until a stop codon is reached. A proportion of the ribosomes slip back by one nucleotide before continuing to translate the message in a different reading frame. Because only a proportion of ribosomes undergo frameshifting at each slippery sequence, there is a gradient of translation from the reading frames at the 5' end of the mRNA to those at the 3' end.
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The slippery sequence alone results in a low frequency of frameshifting. Downstream of the slippery sequence there is an inverted repeat which allows the formation of a stemloop structure.
An additional sequence complementary to the loop allows base-pairing between these two regions, allowing the formation of an RNA pseudoknot. This secondary structure in the mRNA causes ribosomes translating the message to pause at the position of the slippery sequence, increasing the frequency at which frameshifting occurs.
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Termination Suppression
In some retroviruses, the pro gene is separated from the gag gene by a UAG termination codon rather than a 'slippery sequence' and pseudoknot. In the majority of cases, translation of the mRNA terminates at this sequence, giving rise to the gag proteins. In a few instances, the UAG stop codon is suppressed and translation continues, producing a gag-pro-pol polyprotein, which subsequently cleaves itself to produce the mature proteins.
Summary
Control of gene expression is a vital part of virus replication. Co-ordinate expression of groups of virus genes results in successive phases of gene expression. Typically, immediate early genes encode 'activator' proteins, early genes encode further regulatory proteins and late genes encode virus structural proteins. Viruses rely on specific cis- and trans-acting mechanisms to manipulate the biology of their host cells and to enhance and co-ordinate the expression of their own genetic information.

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Chapter 5: Expression

Explain how the pattern of gene expression of a

Expression of Genetic Information

tight control of gene expression.

Expression of Genetic Information

reading frames are commonplace

Control of Prokaryote Gene Expression

Control of Gene Expression in Bacteriophage l

A Simplified Genetic Map of l

Gene Expression in l (1)

(approximately 50 bp) which lies between the cI and N

Gene Expression in l (2)

(approximately 50 bp) which lies between the cI and cro

repressor protein of 236 amino acids which binds to OR,

other genes in the left-hand end of the genome.

Gene Expression in l (3)

OR, blocking binding of the repressor to this site.

Q is an antiterminator similar to N, but only permits

Control of Eukaryote Gene Expression

complex than in prokaryotic cells, a 'multilayered'

forming complicated and dynamic complexes with

compared with the frequency of methylation in

Control of Eukaryote Gene Expression

Control of Eukaryote Gene Expression

Genome Coding Strategies: Class I: Double-Stranded DNA

Polyomaviruses and Papillomaviruses

Herpesvirus Gene Expression

Genome Coding Strategies: Class II: Single-Stranded DNA

Genome Coding Strategies: Class II: SingleStranded DNA

Genome Coding Strategies: Class II: Single-Stranded DNA

Genome Coding Strategies: Class III: Double-Stranded RNA

from their host cells.

Genome Coding Strategies: Class III: DoubleStranded RNA

Genome Coding Strategies: Class III: Double-Stranded RNA

Genome Coding Strategies: Class IV: Single-Stranded (+)Sense RNA

There are two main strategies of gene expression.

Genome Coding Strategies: Class V: Single-Stranded (-)Sense RNA

Packaging of a virus-specific transcriptase/replicase within the virus nucleocapsid is essential.

Genome Coding Strategies: Class V: Single-Stranded (-)Sense RNA

Genome Coding Strategies: Class V: Single-Stranded (-)Sense RNA

Paramyxovirus Gene Expression

Class VI: Single-Stranded (+)Sense RNA with a DNA Intermediate

Class VII: Double-Stranded DNA with an RNA Intermediate

Transcriptional Control of Gene Expression

The SV40 Genome

SV40 Gene Expression

Retrovirus Gene Expression

DNA Binding proteins and Retrovirus LTRs

Retrovirus Gene Expression

These proteins act to increase transcription from

Gene Expression in HTLV and HIV

Control of HTLV Gene Expression

(cyclic AMP response element) sequences in the virus

transcription factors, e.g. p105, a precursor of NF-kB.

Dimerization of these proteins is required for DNA

Control of HIV Gene Expression

Control of HTLV/HIV Gene Expression

Adenovirus Gene Expression

differential splicing of precursor hnRNA transcripts.

regulatory proteins comparable to the tax and tat

Adenovirus Gene Expression

Expression of Adenovirus E1A

Post-Transcriptional Control of Expression by Adenoviruses

Post-Transcriptional Control of Expression by Adenoviruses